The
Diels–Alder reaction is a cornerstone of modern organic
synthesis. Despite this, it remains essentially inaccessible to biosynthetic
approaches. Only a few natural enzymes catalyze even a formal [4 +
2] cycloaddition, and it remains uncertain if any of them proceed
via the Diels–Alder mechanism. In this study, we focus on the
[4 + 2] cycloaddition step in the biosynthesis of spinosyn A, a reaction
catalyzed by SpnF enzyme, one of the most promising “true Diels–Alderase”
candidates. The four currently proposed mechanisms (including the
Diels–Alder one) for this reaction in water (as a first-order
approximation of the enzymatic reaction) are evaluated by an exhaustive
quantum mechanical search for possible transition states (728 were
found in total). We find that the line between the recently proposed
bis-pericyclic [<i>J. Am. Chem. Soc.</i> <b>2016</b>, <i>138</i> (11), 3631] and Diels–Alder routes
is blurred, and favorable transition states of both types may coexist.
Application of the Curtin–Hammett principle, however, reveals
that the bis-pericyclic mechanism accounts for ∼83% of the
reaction flow in water, while the classical Diels–Alder mechanism
contributes only ∼17%. The current findings provide a route
for modeling this reaction inside the SpnF active site and inferring
the catalytic architecture of possible Diels–Alderases.